SUMMARY Influenza is a major public health risk. The current seasonal influenza vaccine is effective against closely matched viruses in healthy adults, but it cannot prevent the outbreaks of epidemics or pandemics because influenza viruses mutate frequently and zoonotic strains can jump the species barrier into humans. Other disadvantages of the seasonal influenza vaccine include the need to produce new vaccines every season, the uncertainty in selecting vaccine strains, and the compromised efficacy for mismatched viruses. A novel generation of influenza vaccines, termed universal influenza vaccines, will overcome these challenges. In the previous grant period, we have produced layered protein nanoparticles (nanoclusters) from conserved HA stalk antigens and the M2 protein ectodomain of influenza A. Nanocluster immunizations induced cross protection against viruses from both phylogenic groups of influenza A, including pandemic-potential avian strains. Both influenza A and influenza B cause influenza epidemics in humans. In this proposal, we propose to construct a multivalent layered nanocluster formulation composed of newly designed antigenic proteins from both influenza A and influenza B as a universal influenza vaccine. The new vaccine will induce broad cross- protection against both influenza types. We have three specific aims: Aim 1. To design and construct conserved antigens from influenza A and B, fabricate nanoclusters from these and previously designed antigenic proteins, and characterize these new nanoclusters. We will optimize the orchestration, composition, and stability of these nanoclusters for the physiologically-activated release of free antigenic proteins, antigen-processing and presentation after the uptake by dendritic cells, distribution of these nanoclusters to draining lymph nodes, and induction of strong antigen-specific immune responses in mice. Aim 2. To test whether these layered nanoclusters or an optimal combination will induce broadly reactive immune responses and whether the immunity will grant cross-protection against viruses spanning both influenza A and influenza B in mice. Aim 3. To test whether the leading multivalent nanocluster combinations will induce robust immune responses which confer broad cross-protection in ferrets. Overall, our research will develop a broadly cross-protective universal influenza vaccine.